Enhanced Diagnostic Tools
Figure 1: Operation of molecular beacons: On their own, these molecules are non-fluorescent, because the stem hybrid keeps the fluorophore close to the quencher. When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence.
In order to detect multiple targets in the same solution, molecular beacons can be made in many different colors utilizing a broad range of fluorophores. DABCYL, a non-fluorescent chromophore, serves as the universal quencher for any fluorophore in molecular beacons. Owing to their stem, the recognition of targets by molecular beacons is so specific that single-nucleotide differences can be readily detected.
Hairpin Tm: The Hairpin stem TM is based on free energy stabilization and folding, the following is a good guideline.
Melting temperatures of G-C rich stem sequences
5 bp = 55 °C to 60 °C
6 bp = 60 °C to 65 °C
7 bp = 65 °C to 70 °C
Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization, Nature Biotechnology 1996; 14: 303-308.
Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination, Nature Biotechnology 1998; 16: 49-53.
Marras SAE, Kramer FR, and Tyagi S (2003) Genotyping single nucleotide polymorphisms with molecular beacons. In Kwok, P. Y. (ed.), Single nucleotide polymorphisms: methods and protocols. The Humana Press Inc., Totowa, NJ, Vol. 212, pp. 111-128. PDF
Vet, J.A.M. and Marras, S.A.E. (2004) Design and optimization of molecular beacon real-time polymerase chain reaction assays. In Herdewijn, P. (ed.), Oligonucleotide synthesis: Methods and Applications. Humana Press, Totowa, NJ, Vol. 288, pp. 273-290.
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